Expression, purification, characterization, and cytotoxic evaluation of the ML1-STxB fusion protein.

Targeted delivery of a toxin substance to cancer cells is one of the most recent cancer treatment options. Mistletoe Lectin-1 (ML1) in Viscum album L. is a Ribosome-inactivating proteins with anticancer properties. Therefore, it appears that a recombinant protein with selective permeability can be generated by fusing ML1 protein with Shiga toxin B, which can bind to Gb3 receptor that is abundantly expressed on cancer cells. In this study, we sought to produce and purify a fusion protein containing ML1 fused to STxB and evaluate its cytotoxic activities. The ML1-STxB fusion protein coding sequence was cloned into the pET28a plasmid, then was transformed into E. coli BL21-DE3 cells. Following induction of protein expression, Ni-NTA affinity chromatography was used to purify the protein. Using SDS-PAGE and western blotting, the expression and purification processes were validated. On the SkBr3 cell line, the cytotoxic effects of the recombinant proteins were evaluated. On SDS-PAGE and western blotting membrane, analysis of purified proteins revealed a band of approximately 41 kDa for rML1-STxB. Ultimately, statistical analysis demonstrated that rML1-STxB exerted significant cytotoxic effects on SkBr3 cells at 18.09 and 22.52 ng/L. The production, purification, and encapsulation of rML1-STxB fusion protein with potential cancer cell-specific toxicity were successful. However, additional research must be conducted on the cytotoxic effects of this fusion protein on other malignant cell lines and in vivo cancer models.

Shiga Toxin, Stx2e, Influences the Activity of Porcine Lymphocytes In Vitro.

Oedema disease (OD) in piglets is one of the most important pathologies, as it causes significant losses due to the high mortality because of the Shiga toxin family, which produces Escherichia coli (STEC) strains. The main toxin responsible for the characteristic pathologies in pigs is Shiga toxin 2 subtype e (Stx2e). Moreover, there is growing evidence that Stx's family of toxins also targets immune cells. Therefore, this study evaluated the effect of different concentrations of Stx2e on porcine immune cells. Porcine peripheral blood mononuclear cells were pre-incubated with Stx2e, at three different concentrations (final concentrations of 10, 500, and 5000 CD50/mL) and with a negative control group. Cells were then stimulated with polyclonal mitogens: concanavalin A, phytohemagglutinin, pokeweed mitogen, or lipopolysaccharides. Cell proliferation was assessed by BrdU (or EdU) incorporation into newly created DNA. The activation of the lymphocyte subsets was assessed by the detection of CD25, using flow cytometry. The toxin significantly decreased mitogen-driven proliferation activity, and the effect was partially dose-dependent, with a significant impact on both T and B populations. The percentage of CD25+ cells was slightly lower in the presence of Stx2e in all the defined T cell subpopulations (CD4+, CD8+, and γδTCR+)-in a dose-dependent manner. B cells seemed to be the most affected populations. The negative effects of different concentrations of Stx2e on the immune cells in this study may explain the negative impact of the subclinical course of OD.

Anomalous Oligomerization Behavior of E. coli Aquaporin Z in Detergent and in Nanodiscs.

Aquaporins are tetrameric integral membrane proteins that act as water channels, and can also permeabilize membranes to other solutes. The monomer appears to be the functional form despite all aquaporins being organized as tetramers, which therefore must provide a clear functional advantage. In addition to this quaternary organization, some aquaporins can act as adhesion molecules in membrane junctions, when tetramers located in opposing membranes interact via their extracellular domains. These stacked forms have been observed in a range of aquaporins, whether using lipidic membrane environments, in electron crystallography, or using detergent micelles, in single-particle cryo-electron microscopy (cryo-EM). In the latter technique, structural studies can be performed when the aquaporin is reconstituted into nanodiscs of lipids that are surrounded by a protein scaffold. During attempts to study E. coli Aquaporin Z (AqpZ), we have found that in some conditions these nanodiscs tend to form filaments that appear to be either thicker head-to-tail or thinner side-to-side stacks of nanodiscs. Nanodisc oligomerization was observed using orthogonal analytical techniques analytical ultra-centrifugation and mass photometry, although the nature of the oligomers (head-to-tail or side-to-side) could not be determined. Using the latter technique, the AqpZ tetramer itself formed oligomers of increasing size when solubilized only in detergent, which is consistent with multiple stacking of AqpZ tetramers. We observed images consistent with both of these filaments in negative staining EM conditions, but only thicker filaments in cryo-EM conditions. We hypothesize that the apparent nanodisc side-to-side arrangement that can only be visualized in negative staining conditions is related to artifacts due to the sample preparation. Filaments of any kind were not observed in EM when nanodiscs did not contain AqpZ, or after addition of detergent into the nanodisc cryo-EM preparation, at concentrations that did not disrupt nanodisc formation. To our knowledge, these filaments have not been observed in nanodiscs preparations of other membrane proteins. AqpZ, like other aquaporins has a charge asymmetry between the cytoplasmic (more positive) and the extracellular sides, which may explain the likely head-to-tail stacking observed, both in nanodisc preparations and also in detergent micelles.

Structure and Assembly of the Proteus mirabilis Flagellar Motor by Cryo-Electron Tomography.

Proteus mirabilis is a Gram-negative Gammaproteobacterium and a major causative agent of urinary tract infections in humans. It is characterized by its ability to switch between swimming motility in liquid media and swarming on solid surfaces. Here, we used cryo-electron tomography and subtomogram averaging to reveal the structure of the flagellar motor of P. mirabilis at nanometer resolution in intact cells. We found that P. mirabilis has a motor that is structurally similar to those of Escherichia coli and Salmonella enterica, lacking the periplasmic elaborations that characterize other more specialized gammaproteobacterial motors. In addition, no density corresponding to stators was present in the subtomogram average suggesting that the stators are dynamic. Finally, several assembly intermediates of the motor were seen that support the inside-out assembly pathway.

Mechanism of ADP-Inhibited ATP Hydrolysis in Single Proton-Pumping FoF1-ATP Synthase Trapped in Solution.

FoF1-ATP synthases in mitochondria, in chloroplasts, and in most bacteria are proton-driven membrane enzymes that supply the cells with ATP made from ADP and phosphate. Different control mechanisms exist to monitor and prevent the enzymes' reverse chemical reaction of fast wasteful ATP hydrolysis, including mechanical or redox-based blockade of catalysis and ADP inhibition. In general, product inhibition is expected to slow down the mean catalytic turnover. Biochemical assays are ensemble measurements and cannot discriminate between a mechanism affecting all enzymes equally or individually. For example, all enzymes could work more slowly at a decreasing substrate/product ratio, or an increasing number of individual enzymes could be completely blocked. Here, we examined the effect of increasing amounts of ADP on ATP hydrolysis of single Escherichia coli FoF1-ATP synthases in liposomes. We observed the individual catalytic turnover of the enzymes one after another by monitoring the internal subunit rotation using single-molecule Förster resonance energy transfer (smFRET). Observation times of single FRET-labeled FoF1-ATP synthases in solution were extended up to several seconds using a confocal anti-Brownian electrokinetic trap (ABEL trap). By counting active versus inhibited enzymes, we revealed that ADP inhibition did not decrease the catalytic turnover of all FoF1-ATP synthases equally. Instead, increasing ADP in the ADP/ATP mixture reduced the number of remaining active enzymes that operated at similar catalytic rates for varying substrate/product ratios.

Yields and product comparison between Escherichia coli BL21 and W3110 in industrially relevant conditions: anti-c-Met scFv as a case study.

INTRODUCTION: In the biopharmaceutical industry, Escherichia coli is one of the preferred expression hosts for large-scale production of therapeutic proteins. Although increasing the product yield is important, product quality is a major factor in this industry because greatest productivity does not always correspond with the highest quality of the produced protein. While some post-translational modifications, such as disulphide bonds, are required to achieve the biologically active conformation, others may have a negative impact on the product's activity, effectiveness, and/or safety. Therefore, they are classified as product associated impurities, and they represent a crucial quality parameter for regulatory authorities. RESULTS: In this study, fermentation conditions of two widely employed industrial E. coli strains, BL21 and W3110 are compared for recombinant protein production of a single-chain variable fragment (scFv) in an industrial setting. We found that the BL21 strain produces more soluble scFv than the W3110 strain, even though W3110 produces more recombinant protein in total. A quality assessment on the scFv recovered from the supernatant was then performed. Unexpectedly, even when our scFv is correctly disulphide bonded and cleaved from its signal peptide in both strains, the protein shows charge heterogeneity with up to seven distinguishable variants on cation exchange chromatography. Biophysical characterization confirmed the presence of altered conformations of the two main charged variants. CONCLUSIONS: The findings indicated that BL21 is more productive for this specific scFv than W3110. When assessing product quality, a distinctive profile of the protein was found which was independent of the E. coli strain. This suggests that alterations are present in the recovered product although the exact nature of them could not be determined. This similarity between the two strains' generated products also serves as a sign of their interchangeability. This study encourages the development of innovative, fast, and inexpensive techniques for the detection of heterogeneity while also provoking a debate about whether intact mass spectrometry-based analysis of the protein of interest is sufficient to detect heterogeneity in a product.

Mapping of RNase P Ribozyme Regions in Proximity with a Human RNase P Subunit Protein Using Fe(II)-EDTA Cleavage and Nuclease Footprint Analyses.

Ribonuclease P (RNase P), which may consist of both protein subunits and a catalytic RNA part, is responsible for 5' maturation of tRNA by cleaving the 5'-leader sequence. In Escherichia coli, RNase P contains a catalytic RNA subunit (M1 RNA) and a protein factor (C5 protein). In human cells, RNase P holoenzyme consists of an RNA subunit (H1 RNA) and multiple protein subunits that include human RPP29 protein. M1GS, a sequence specific targeting ribozyme derived from M1 RNA, can be constructed to target a specific mRNA to degrade it in vitro. Recent studies have shown that M1GS ribozymes are efficient in blocking the expression of viral mRNAs in cultured cells and in animals. These results suggest that RNase P ribozymes have the potential to be useful in basic research and in clinical applications. It has been shown that RNase P binding proteins, such as C5 protein and RPP29, can enhance the activities of M1GS RNA in processing a natural tRNA substrate and a target mRNA. Understanding how RPP29 binds to M1GS RNA and enhances the enzyme's catalytic activity will provide great insight into developing more robust gene-targeting ribozymes for in vivo application. In this chapter, we describe the methods of using Fe(II)-ethylenediaminetetraacetic acid (EDTA) cleavage and nuclease footprint analyses to determine the regions of a M1GS ribozyme that are in proximity to RPP29 protein.

MS2-MBP-Based Affinity Purification of Nucleus- or Cytoplasm-Localized lncRNA-Protein Complexes Formed In Vivo.

With recent emergence of huge number of long noncoding RNAs (lncRNAs), purification of lncRNA-protein (lncRNP) complexes is fundamental to understand the role of lncRNA and its biological function. However, lncRNP purification is still a daunting challenge. Here we describe a protocol to purify lncRNP formed in vivo with MS2-MBP-based affinity purification. Inducible lncRNA tagged with MS2 RNA hairpins is introduced into cells of interest, and RNP on tagged lncRNA is formed in vivo. MS2-MBP fusion protein is expressed in Escherichia coli and purified with amylose resin and HiTrap heparin column. The MS2 part of MS2-MBP fusion protein binds to the hairpins, and MBP part binds to amylose resin. We also describe a protocol to separate the nucleus and the cytoplasm so that lncRNP localized in the nucleus or cytoplasm can be individually purified. The amount of lncRNP purified is well sufficient for mass spectrometry analysis.

A synthetic population-level oscillator in non-microfluidic environments.

Synthetic oscillators have become a research hotspot because of their complexity and importance. The construction and stable operation of oscillators in large-scale environments are important and challenging. Here, we introduce a synthetic population-level oscillator in Escherichia coli that operates stably during continuous culture in non-microfluidic environments without the addition of inducers or frequent dilution. Specifically, quorum-sensing components and protease regulating elements are employed, which form delayed negative feedback to trigger oscillation and accomplish the reset of signals through transcriptional and post-translational regulation. We test the circuit in devices with 1 mL, 50 mL, 400 mL of medium, and demonstrate that the circuit could maintain stable population-level oscillations. Finally, we explore potential applications of the circuit in regulating cellular morphology and metabolism. Our work contributes to the design and testing of synthetic biological clocks that function in large populations.

An industrially applicable Escherichia coli platform for bioconversion of thebaine to oripavine and codeine to morphine.

A whole cell Escherichia coli biotransformation platform converting thebaine to oripavine and codeine to morphine was demonstrated with industrially applicable yields (∼1.2 × 10-2 g L-1 h-1 or ∼1.2 × 10-1 g L-1 h-1), improving >13 400-fold upon morphine production in yeast. Mutations enhanced enzyme performance and the use of a purified substrate with rich raw poppy extract expanded applicability.

Phenotypic and Genotypic Adaptation of Escherichia coli to Thermal Stress is Contingent on Genetic Background.

Evolution can be contingent on history, but we do not yet have a clear understanding of the processes and dynamics that govern contingency. Here, we performed the second phase of a two-phase evolution experiment to investigate features of contingency. The first phase of the experiment was based on Escherichia coli clones that had evolved at the stressful temperature of 42.2 °C. The Phase 1 lines generally evolved through two adaptive pathways: mutations of rpoB, which encodes the beta subunit of RNA polymerase, or through rho, a transcriptional terminator. We hypothesized that epistatic interactions within the two pathways constrained their future adaptative potential, thus affecting patterns of historical contingency. Using ten different E. coli Founders representing both adaptive pathways, we performed a second phase of evolution at 19.0 °C to investigate how prior genetic divergence or adaptive pathway (rpoB vs. rho) affects evolutionary outcomes. We found that phenotype, as measured by relative fitness, was contingent on founder genotypes and pathways. This finding extended to genotypes, because E. coli from different Phase 1 histories evolved by adaptive mutations in distinct sets of genes. Our results suggest that evolution depends critically on genetic history, likely due to idiosyncratic epistatic interactions within and between evolutionary modules.

Purification of recombinant bacterial collagens containing structural perturbations.

Streptococcus pyogenes-derived recombinant bacterial collagen-like proteins (CLPs) are emerging as a potential biomaterial for biomedical research and applications. Bacterial CLPs form stable triple helices and lack specific interactions with human cell surface receptors, thus enabling the design of novel biomaterials with specific functional attributes. Bacterial collagens have been instrumental in understanding collagen structure and function in normal and pathological conditions. These proteins can be readily produced in E. coli, purified using affinity chromatography, and subsequently isolated after cleavage of the affinity tag. Trypsin is a widely used protease during this purification step since the triple helix structure is resistant to trypsin digestion. However, the introduction of Gly→X mutations or natural interruptions within CLPs can perturb the triple helix structure, making them susceptible to trypsin digestion. Consequently, removing the affinity tag and isolating collagen-like (CL) domains containing mutations is impossible without degradation of the product. We present an alternative method to isolate CL domains containing Gly→X mutations utilizing a TEV protease cleavage site. Protein expression and purification conditions were optimized for designed protein constructs to achieve high yield and purity. Enzymatic digestion assays demonstrated that CL domains from wild-type CLPs could be isolated by digestion with either trypsin or TEV protease. In contrast, CLPs containing Gly→Arg mutations are readily digested by trypsin while digestion with TEV protease cleaved the His6-tag, enabling the isolation of mutant CL domains. The developed method can be adapted to CLPs containing various new biological sequences to develop multifunctional biomaterials for tissue engineering applications.

L-Lactate treatment by photosynthetic cyanobacteria expressing heterogeneous L-lactate dehydrogenase.

L-Lactate is a major waste compound in cultured animal cells. To develop a sustainable animal cell culture system, we aimed to study the consumption of L-lactate using a photosynthetic microorganism. As genes involved in L-lactate utilization were not found in most cyanobacteria and microalgae, we introduced the NAD-independent L-lactate dehydrogenase gene from Escherichia coli (lldD) into Synechococcus sp. PCC 7002. The lldD-expressing strain consumed L-lactate added to basal medium. This consumption was accelerated by expression of a lactate permease gene from E. coli (lldP) and an increase in culture temperature. Intracellular levels of acetyl-CoA, citrate, 2-oxoglutarate, succinate, and malate, and extracellular levels of 2-oxoglutarate, succinate, and malate, increased during L-lactate utilization, suggesting that the metabolic flux from L-lactate was distributed toward the tricarboxylic acid cycle. This study provides a perspective on L-lactate treatment by photosynthetic microorganisms, which would increase the feasibility of animal cell culture industries.

Gut microbiome of mealworms (Tenebrio molitor Larvae) show similar responses to polystyrene and corn straw diets.

BACKGROUND: Some insects can degrade both natural and synthetic plastic polymers, their host and gut microbes play crucial roles in this process. However, there is still a scientific gap in understanding how the insect adapted to the polystyrene (PS) diet from natural feed. In this study, we analyzed diet consumption, gut microbiota responses, and metabolic pathways of Tenebrio molitor larvae exposed to PS and corn straw (CS). RESULTS: T. molitor larvae were incubated under controlled conditions (25 ± 1 °C, 75 ± 5% humidity) for 30 days by using PS foam with weight-, number-, and size-average molecular weight (Mw, Mn, and Mz) of 120.0, 73.2, and 150.7 kDa as a diet, respectively. The larvae exhibited lower PS consumption (32.5%) than CS (52.0%), and these diets had no adverse effects on their survival. The gut microbiota structures, metabolic pathways, and enzymatic profiles of PS- and CS-fed larvae showed similar responses. The gut microbiota of larvae analysis indicated Serratia sp., Staphylococcus sp., and Rhodococcus sp. were associated with both PS and CS diets. Metatranscriptomic analysis revealed that xenobiotics, aromatic compounds, and fatty acid degradation pathways were enriched in PS- and CS-fed groups; laccase-like multicopper oxidases, cytochrome P450, monooxygenase, superoxidase, and dehydrogenase were involved in lignin and PS degradation. Furthermore, the upregulated gene lac640 in both PS- and CS-fed groups was overexpressed in E. coli and exhibited PS and lignin degradation ability. CONCLUSIONS: The high similarity of gut microbiomes adapted to biodegradation of PS and CS indicated the plastics-degrading ability of the T. molitor larvae originated through an ancient mechanism that degrades the natural lignocellulose. Video Abstract.

Structural and functional features of a broad-spectrum prophage-encoded enzybiotic from Enterococcus faecium.

Multidrug-resistant (MDR) bacteria have become a growing threat to public health. The gram-positive Enterococcus faecium is classified by WHO as a high-priority pathogen among the global priority list of antibiotic-resistant bacteria. Peptidoglycan-degrading enzymes (PDEs), also known as enzybiotics, are useful bactericidal agents in the fight against resistant bacteria. In this work, a genome-based screening approach of the genome of E. faecium allowed the identification of a putative PDE gene with predictive amidase activity (EfAmi1; EC 3.5.1.28) in a prophage-integrated sequence. EfAmi1 is composed by two domains: a N-terminal Zn2+-dependent N-acetylmuramoyl-L-alanine amidase-2 (NALAA-2) domain and a C-terminal domain with unknown structure and function. The full-length gene of EfAmi1 was cloned and expressed as a 6xHis-tagged protein in E. coli. EfAmi1 was produced as a soluble protein, purified, and its lytic and antimicrobial activities were investigated using turbidity reduction and Kirby-Bauer disk-diffusion assays against clinically isolated bacterial pathogens. The crystal structure of the N-terminal amidase-2 domain was determined using X-ray crystallography at 1.97 Å resolution. It adopts a globular fold with several α-helices surrounding a central five-stranded ß-sheet. Sequence comparison revealed a cluster of conserved amino acids that defines a putative binding site for a buried zinc ion. The results of the present study suggest that EfAmi1 displays high lytic and antimicrobial activity and may represent a promising new antimicrobial in the post-antibiotic era.

Biotechnological product potential of Auxenochlorella protothecoides including biologically active compounds (BACs) under nitrogen stress conditions.

Nitrogen stress can influence microalgae's growth characteristics, and microalgae grown in nitrogen-deficient conditions may produce higher or lower levels of biotechnological products as a result of metabolic changes. In photoautotrophic and heterotrophic cultures, nitrogen limitation has been proven effective in promoting lipid accumulation. In spite of this, no study has demonstrated a significant correlation between lipid content and other biotechnological products such as bioactive compounds (BACs). This research examines a strategy for lipid accumulation as well as the potential production of BACs with antibacterial properties in parallel with that strategy. This concept involved the treatment of the microalga Auxenochlorella protothecoides with low and high concentrations of ammonium (NH4+). This particular experiment reached a maximum lipid content of 59.5% using a 0.8 mM NH4+ concentration, resulting in the yellowing of the chlorophyll levels. Agar diffusion assays were conducted to determine the antibacterial activity of different extracts derived from the biomass when stressed with different levels of nitrogen. Algal extracts prepared by a variety of solvents showed different levels of antibacterial activity against representative strains of both gram-negative (Escherichia coli) and gram-positive (Staphylococcus aureus) bacteria. Among the extracts tested, 500 mg/L ethyl acetate extract had the greatest antibacterial activity against Escherichia coli. In order to identify the components responsible for the extract's antibacterial activity, fatty acid methyl ester (FAME) analysis was performed. It has been suggested that the lipid fraction may be a valuable indicator of these activities since some lipid components are known to possess antimicrobial properties. In this regard, it was found that the amount of polyunsaturated fatty acid (PUFA) significantly decreased by 53.4% under the conditions with the highest antibacterial activity observed.

[Synergistic effect of ß-thujaplicin and tigecycline against tet(X4)-positive Escherichia coli in vitro].

The widespread of tigecycline resistance gene tet(X4) has a serious impact on the clinical efficacy of tigecycline. The development of effective antibiotic adjuvants to combat the looming tigecycline resistance is needed. The synergistic activity between the natural compound ß-thujaplicin and tigecycline in vitro was determined by the checkerboard broth microdilution assay and time-dependent killing curve. The mechanism underlining the synergistic effect between ß-thujaplicin and tigecycline against tet(X4)-positive Escherichia coli was investigated by determining cell membrane permeability, bacterial intracellular reactive oxygen species (ROS) content, iron content, and tigecycline content. ß-thujaplicin exhibited potentiation effect on tigecycline against tet(X4)-positive E. coli in vitro, and presented no significant hemolysis and cytotoxicity within the range of antibacterial concentrations. Mechanistic studies demonstrated that ß-thujaplicin significantly increased the permeability of bacterial cell membranes, chelated bacterial intracellular iron, disrupted the iron homeostasis and significantly increased intracellular ROS level. The synergistic effect of ß-thujaplicin and tigecycline was identified to be related to interfere with bacterial iron metabolism and facilitate bacterial cell membrane permeability. Our studies provided theoretical and practical data for the application of combined ß-thujaplicin with tigecycline in the treatment of tet(X4)-positive E. coli infection.

[Expression, purification, and characterization of the histidine kinase CarS from Fusobacterium nucleatum].

Fusobacterium nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues, affecting multiple stages of colorectal cancer development. The two-component system plays an important role in the regulation and expression of genes related to pathogenic resistance and pathogenicity. In this paper, we focused on the CarRS two-component system of F. nucleatum, and the histidine kinase protein CarS was recombinantly expressed and characterized. Several online software such as SMART, CCTOP and AlphaFold2 were used to predict the secondary and tertiary structure of the CarS protein. The results showed that CarS is a membrane protein with two transmembrane helices and contains 9 α-helices and 12 ß-folds. CarS protein is composed of two domains, one is the N-terminal transmembrane domain (amino acids 1-170), the other is the C-terminal intracellular domain. The latter is composed of a signal receiving domain (histidine kinases, adenylyl cyclases, methyl-accepting proteins, prokaryotic signaling proteins, HAMP), a phosphate receptor domain (histidine kinase domain, HisKA), and a histidine kinase catalytic domain (histidine kinase-like ATPase catalytic domain, HATPase_c). Since the full-length CarS protein could not be expressed in host cells, a fusion expression vector pET-28a(+)-MBP-TEV-CarScyto was constructed based on the characteristics of secondary and tertiary structures, and overexpressed in Escherichia coli BL21-Codonplus(DE3)RIL. CarScyto-MBP protein was purified by affinity chromatography, ion-exchange chromatography, and gel filtration chromatography with a final concentration of 20 mg/ml. CarScyto-MBP protein showed both protein kinase and phosphotransferase activities, and the MBP tag had no effect on the function of CarScyto protein. The above results provide a basis for in-depth analysis of the biological function of the CarRS two-component system in F. nucleatum.

Metabolic Engineering of Escherichia coli for the Biosynthesis of 3-Phenylpropionic Acid and 3-Phenylpropyl Acetate.

3-Phenylpropionic acid (3PPA) and its derivative 3-phenylpropyl acetate (3PPAAc) are important aromatic compounds with broad applications in the cosmetics and food industries. In this study, we constructed a plasmid-free 3PPA-producing Escherichia coli strain and designed a novel 3PPAAc biosynthetic pathway. A module containing tyrosine ammonia lyase and enoate reductase, evaluated under the control of different promoters, was combined with phenylalanine-overproducing strain E. coli ATCC31884, enabling the plasmid-free de novo production of 218.16 ± 43.62 mg L-1 3PPA. The feasibility of the pathway was proved by screening four heterologous alcohol acetyltransferases, which catalyzed the transformation of 3-phenylpropyl alcohol into 3PPAAc. Afterward, 94.59 ± 16.25 mg L-1 3PPAAc was achieved in the engineered E. coli strain. Overall, we have not only demonstrated the potential of de novo synthesis of 3PPAAc in microbes for the first time but also provided a platform for the future of biosynthesis of other aromatic compounds.

Using NMR Titration Experiments to Study E. coli FAS-II- and AcpP-Mediated Protein-Protein Interactions.

Acyl carrier proteins (ACPs) are central to many primary and secondary metabolic pathways. In E. coli fatty acid biosynthesis (FAB), the central ACP, AcpP, transports intermediates to a suite of partner proteins (PP) for iterative modification and elongation. The regulatory protein-protein interactions that occur between AcpP and the PP in FAB are poorly understood due to the dynamic and transient nature of these interactions. Solution-state NMR spectroscopy can reveal information at the atomic level through experiments such as the 2D heteronuclear single quantum coherence (HSQC). The following protocol describes NMR HSQC titration experiments that can elucidate biomolecular recognition events.